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A K Hajra

Publications and source records attributed to A K Hajra.

At least 19 recordsLinked to original sources
PubMed2000

Molecular cloning and expression of mammalian peroxisomal trans-2-enoyl-coenzyme A reductase cDNAs.

Chain elongation of fatty acids is an important cellular process and is believed to occur in the endoplasmic reticulum of all eukaroytic cells. Herein we describe the cloning and characterization of a peroxisomal NADPH-specific trans-2-enoyl-CoA reductase, the key enzyme for a proposed peroxisomal chain elongation pathway. The reductase was solubilized and partially purified from guinea pig liver peroxisomes by affinity chromatography. On SDS-polyacrylamide gel electrophoresis, a 40-kDa band was identified as the enzyme, and its partial amino acid sequence (27 amino acids) was determined. A full-length cDNA for the reductase was cloned from a guinea pig liver cDNA library. The open reading frame of this nucleotide sequence encodes a 302-amino acid polypeptide with a calculated molecular mass of 32.5 kDa. Full-length mouse and human cDNA clones encoding homologous proteins have also been isolated. All of these translated polypeptides have the type I peroxisomal targeting signal, AKL, at the carboxyl terminus. The identity of the cloned enoyl-CoA reductase cDNAs was confirmed by expressing the guinea pig and human cDNAs in Escherichia coli. The His-tagged recombinant enzymes were found to have very high NADPH-specific 2-enoyl-CoA reductase activity with similar properties and specificity as the liver peroxisomal reductase. Both the natural and the recombinant enzyme catalyze the reduction of trans-2-enoyl-CoAs of varying chain lengths from 6:1 to 16:1, having maximum activity with 10:1 CoA. Northern blot analysis demonstrated that a single transcript of 1.3 kilobases is present in most mouse tissues, with particularly high concentrations in liver and kidney.

Amino Acid Sequence↗
PubMed2000

Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis.

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using D-[3-(3)H, U-(14)C]glucose as the precursor of the lipid glycerol backbone shows that about 40-50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids.

3T3 Cells↗
PubMed1998

Developmental delay and growth failure caused by a peroxisomal disorder, dihydroxyacetonephosphate acyltransferase (DHAP-AT) deficiency.

We describe a 6 1/2-year-old-girl presenting with a unique phenotype and dihydroxyacetonephosphate acyltransferase (DHAP-AT) deficiency (1.6% of control activity in cultured fibroblasts), a peroxisomal enzyme deficiency which was reported previously to cause rhizomelic chondroplasia punctata (RCDP). Her phenotype is less severe than that seen in classical RCDP, and is notable for short stature, microcataracts, normal limbs, mild hypotonia, and severe mental retardation. Epiphyseal stippling is present. This patient illustrates the variability of peroxisomal disorders whereby a specific defect in peroxisomal plasmalogen synthesis may lead to several phenotypes. Her case also suggests that children presenting with deficient growth, developmental delay, and epiphyseal stippling should be screened carefully for peroxisomal disorders, with measurement of plasmalogens in addition to very long chain fatty acids.

Acyltransferases↗
PubMed1998

Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.

We have developed a two-step selection protocol to generate a population of Chinese hamster ovary (CHO) cell variants that are plasmalogen-deficient, but contain intact, functional peroxisomes (plasmalogen-/peroxisome+). This involved sequential exposures of a mutagenized cell population to photodynamic damage by using two different pyrene-labelled sensors, 9-(1'-pyrene)nonanol and 12-(1'-pyrene)dodecanoic acid. By this procedure we generated several isolates, all except one of which displayed a severe decrease in plasmalogen biosynthesis. Further characterization of one of the plasmalogen-deficient isolates, NRel-4, showed that it contained intact, functional peroxisomes. Whole-cell homogenates from NRel-4 displayed severely decreased dihydroxyacetone phosphate acyltransferase, which catalyses the first step in plasmalogen biosynthesis. NRel-4 and another, recently described, plasmalogen-deficient cell line, NZel-1 [Nagan, Hajra, Das, Moser, Moser, Lazarow, Purdue and Zoeller (1997) Proc. Natl. Acad. Sci. U.S. A. 94, 4475-4480] were hypersensitive to singlet oxygen, supporting the notion of plasmalogens as radical oxygen scavengers. Wild-type-like resistance could be conferred on NRel-4 upon restoration of plasmalogen content by supplementation with a bypass compound, sn-1-hexadecylglycerol. NRel-4 and other plasmalogen-/peroxisome+ strains will allow us to examine further the role of ether lipids in cellular functions without complications associated with peroxisome deficiency, and might serve as an animal cell model for certain forms of the human genetic disorder rhizomelic chondrodysplasia punctata.

Acyltransferases↗
PubMed1998

Enzymatic synthesis of [3H]Cytidine 5'-diphospho-1, 2-diacyl-sn-glycerol.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol; CDP-DG) is an important intermediate in the biosynthesis of the major glycerophosphate-based phospholipids of prokaryotes and eukaryotes. This compound is expensive to purchase and inefficient to prepare chemically. Radiolabeled CDP-diacylglycerol is unavailable commercially. We describe a simple and inexpensive method to synthesize [3H]CDP-DG enzymatically. The three-step enzymatic procedure includes phosphorylation of [3H]glycerol to sn-[3H]glycerol 3-phosphate (G3P) by glycerokinase,acylation of [3H]G3P to [3H]phosphatidic acid (PA) by G3P acyltransferase, and conversion of [3H]PA and CTP to [3H]CDP-DG by CDP-DG synthase. This procedure is considerably less labor intensive and less expensive than is chemical synthesis, and the yield is at least 30%.

Acylation↗
PubMed1997

An animal cell mutant with a deficiency in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Effects on the biosynthesis of ether-linked and diacyl glycerolipids.

In the accompanying paper (James, P. F., and Zoeller, R. A. (1997) J. Biol. Chem. 272, 23532-23539), we reported the isolation of a series of mutants from the fibroblast-like cell line, CHO-K1, that are deficient in the incorporation of the long chain fatty alcohol, hexadecanol, into complex lipids. All but one of these mutants, FAA. K1B, were deficient in long-chain-fatty alcohol oxidase (FAO) activity. We have further characterized this FAO+ isolate. FAA.K1B cells displayed a 40% decrease in [9,10-3H]hexadecanol uptake when compared with the parent strain. Although incorporation of hexadecanol into the phospholipid fraction was decreased by 52%, the cells accumulated label in alkylglycerol (20-fold over wild type). The increase in 1-alkylglycerol labeling corresponded to a 4-fold increase in alkylglycerol mass. Short term labeling with 32Pi showed a 45-50% decrease in overall phospholipid biosynthesis in FAA.K1B. Both diacyl- and ether-linked species were affected, suggesting a general defect in phospholipid biosynthesis. Mutant cells were able to partially compensate for the decreased biosynthesis by decreasing the turnover of the phospholipid pools. The primary lesion in FAA. K1B was identified as a 95% reduction in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Whole cell homogenates from FAA.K1B were unable to reduce either acyl-dihydroxyacetone phosphate (DHAP) or alkyl-DHAP, supporting the notion that the reduction of these two compounds is catalyzed by a single enzyme. These data suggest that the biosynthesis of diacyl phospholipids, in Chinese hamster ovary cells, begins with the acylation of dihydroxyacetone phosphate as well as glycero-3-phosphate and that the "DHAP pathway" contributes significantly to diacyl glycerolipid biosynthesis. Also, the severe reduction in acyl/alkyl-DHAP reductase activity in FAA.K1B resulted in only a moderate decrease in ether lipid biosynthesis. These latter data together with the observed increase in alkylglycerol levels support the existence of a shunt pathway that is able to partially bypass the enzymatic lesion.

Animals↗
PubMed1997

Dihydroxyacetone phosphate acyltransferase.

In this article the properties, assay, distribution, subcellular localization, deficiency in congenital peroxisomal disorders, purification and physiological functions of dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) are reviewed.

Acyltransferases↗
PubMed1997

A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.

Using fluorescence-activated cytotoxicity selection, followed by colony autoradiographic screening of the surviving population, we have isolated a unique plasmalogen-deficient Chinese hamster ovary (CHO) cell line. The mutant, NZel-1, showed a dramatic (90%) reduction in the rate of biosynthesis and levels of plasmalogens, as determined using short- and long-term labeling with 32Pi. Enzymatic assays and lipid supplementation studies showed that NZel-1 was defective in a single step in the biosynthetic pathway for plasmalogens. This step, catalyzed by the peroxisomal enzyme, alkyl-dihydroxyacetone phosphate (DHAP) synthase, is responsible for the introduction of the ether bond found in plasmalogens. The activity of alkyl-DHAP synthase was reduced in whole-cell homogenates from NZel-1 to 18% of wild-type values. Unlike previously described plasmalogen-deficient mutants, NZel-1 contained peroxisomes, as confirmed by immunofluorescence microscopy and catalase release by digitonin. Peroxisomal functions, including the breakdown of very long-chain (>20 carbons) fatty acids, phytanic acid oxidation, and the acylation of DHAP, were normal. Cell fusion studies revealed that the mutation is recessive and belongs to a new complementation group. To our knowledge this is the first report describing the isolation and characterization of a mutant CHO cell line defective in plasmalogen biosynthesis which contains intact, functional peroxisomes. These cells will allow us to examine the role of ether lipids in cellular functions without complications associated with peroxisome deficiency.

Alkyl and Aryl Transferases↗
PubMed1996

Molecular cloning and expression of cDNA encoding rat brain cytosolic acyl-coenzyme A thioester hydrolase.

The cDNA encoding rat brain cytosolic acyl-CoA thioester hydrolase (ACT) has been cloned and sequenced, and the primary structure of the enzyme has been deduced. A partial amino acid sequence (38 amino acids) of the enzyme was determined using the peptides generated after CNBr digestion of the purified enzyme. Primers synthesized on the basis of this information were used to isolate two cDNA clones, each encoding the full length of the enzyme. The nucleotide sequences of these clones contained an open reading frame encoding a 358-amino acid polypeptide with a calculated molecular mass of 39.7 kDa, similar to that determined for the purified enzyme (40.9 kDa). The deduced ACT sequence showed no homology to the known sequences of any other thioesterases nor to any other known protein sequence. However, there was a strong homology to a number of expressed sequence tag human brain cDNA clones. The identity of the ACT cDNA was confirmed by the expression of ACT activity in Escherichia coli. There was a 10-15-fold increase in ACT-specific activity in the bacterial extracts after induction with isopropyl thiogalactoside, and the properties of the expressed enzyme (fusion protein) were the same as those of the purified rat brain ACT. Northern blot analysis showed that a 1.65-kilobase ACT transcript was present in rat brain and testis but not in any other rat tissues examined. However, the ACT mRNA was induced in the liver of rats that were fed Wy-14,643, a peroxisome proliferator and inducer of rodent liver cytosolic acyl-CoA thioesterase. These results indicate that the induced rat liver ACT is homologous to the constitutive rat brain ACT.

Amino Acid Sequence↗
PubMed1996

A novel chemical synthesis of 1-O-hexadecyl-rac-[2-3H]glycero-3-phosphorylethanolamine and a simple assay for plasmanyl desaturase.

A simple and efficient method for chemical synthesis of lysophosphatidylethanolamine is described. 1-O-hexadecyl diazohydroxyacetone (A. K. Hajra, T. V. Saraswathi and A. K. Das. 1983. Chem. Phys. Lipids. 33: 179-193) was decomposed by benzyloxycarbonyl (CBZ) derivative of phosphorylethanolamine (I) to give 1-O-hexadecyl dihydroxyacetone-3-(N)-CBZ phosphorylethanolamine (II). Compound (II) was reduced by NaBH4 and the product (III), after catalytic transfer hydrogenolysis produced the final compound, 1-O-hexadecyl-rac-glycero-3-phosphorylethanolamine (IV). The yield of (IV), starting from 1-O-hexadecyl diazohydroxyacetone was 53%. The identities of the compounds were verified by NMR and fast atom bombardment mass spectral (FAB-MS) analysis. 1-O-hexadecyl-rac[2-3H]glycero-3-phosphorylethanolamine prepared by the method above was shown to be a good in vitro substrate for plasmanyl delta 1'-desaturase (EC 1.14.99.19). Using this radioactive substrate, a simple and rapid solvent partition assay for this enzyme was developed with results comparable to those obtained by the two-dimensional thin-layer chromatographic assay method. The advantage of this rapid assay system and the applicability of the chemical synthetic method for other phosphoglycerides are discussed.

Biological Assay↗
PubMed1995

Phenotype of patients with peroxisomal disorders subdivided into sixteen complementation groups.

OBJECTIVE: To use the technique of complementation analysis to help define genotype and classify patients with clinical manifestations consistent with those of the disorders of peroxisome assembly, namely the Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). STUDY DESIGN: Clinical findings, peroxisomal function, and complementation groups were examined in 173 patients with the clinical manifestations of these disorders. RESULTS: In 37 patients (21%), peroxisome assembly was intact and isolated deficiencies of one of five peroxisomal enzymes involved in the beta-oxidation of fatty acids or plasmalogen biosynthesis were demonstrated. Ten complementation groups were identified among 93 patients (54%) with impaired peroxisome assembly and one of three phenotypes (ZS, NALD, or IRD) without correlation between complementation group and phenotype. Forty-three patients (25%) had impaired peroxisome assembly associated with the RCDP phenotype and belonged to a single complementation group. Of the 173 patients, 10 had unusually mild clinical manifestations, including survival to the fifth decade or deficits limited to congenital cataracts. CONCLUSIONS: At least 16 complementation groups, and hence genotypes, are associated with clinical manifestations of disorders of peroxisome assembly. The range of phenotype is wide, and some patients have mild involvement.

Acyltransferases↗
PubMed1995

Purification, properties, and specificity of rat brain cytosolic fatty acyl coenzyme A hydrolase.

Rat brain cytosolic acyl-CoA hydrolase has been purified 3,500-fold to apparent homogeneity using heat treatment, ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and hydroxyapatite c chromatography. The purified enzyme remains stable only in the presence of a high concentration (30%, vol/vol) of ethylene glycol. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme shows a single band of 40.9 kDa. However, on high-performance size-exclusion chromatography the migration rate of the enzyme corresponds with an apparent molecular mass of 148 kDa, indicating that the native enzyme may be a tetramer. The enzyme catalyzes the hydrolysis of fatty acyl-CoAs from six to 18 carbon chains long, having the highest activity for lauroyl (12:0)-CoA. For the purified enzyme the Km for palmitoyl-CoA is 5.8 microM and the Vmax is 1,300 mumol/min/mg of protein. The enzyme is inhibited by bovine serum albumin, various detergents, lysophosphatidylcholine, and palmitoyl carnitine. Among the sulfhydryl agents, only p-hydroxymercuribenzoate inhibited the enzyme. The enzyme is also inactivated by treatment with a high concentration of diethyl pyrocarbonate, an active center histidine-reacting agent, but not by phenylmethylsulfonyl fluoride (10 mM), a serine esterase inhibitor. The purified enzyme does not appear to possess any O-ester hydrolase, lysophospholipase, transacylase, or acyltransferase activity.

Amino Acids↗
PubMed1995

A simple chemical synthesis of the ether analog of lysophosphatidylcholine and platelet-activating factor.

A simple chemical procedure for synthesis of 1-O-alkyl-(rac or sn)-glycero-3-phosphocholine (alkyl analog of lysophosphatidylcholine, II) and platelet activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (III) has been described. The key step of the method is the decomposition of 1-O-hexadecyl-3-diazohydroxyacetone (A. K. Hajra, T. V. Saraswathi and A. K. Das, 1983. Chem. Phys. Lipids. 33: 179-193) with phosphocholine to synthesize 1-O-hexadecyl dihydroxyacetone-3-phosphocholine (I). Conditions for this phosphorolysis were studied with respect to the reaction medium, temperature, and optimum proportion of the reactants. Compound (I) was quantitatively reduced with NaBH4 to synthesize (II) which was acetylated to prepare compound (III). Phospholipase A2 hydrolysis of compound (III) followed by separation of the products afforded the unreacted sn-3-hexadecyl isomer (III) and sn-1-hexadecyl isomer (II) which was acetylated to PAF. The structures of the compounds were verified by NMR and FAB-MS spectra, and their biological activities were determined by measuring the release of serotonin from rabbit blood platelets in response to different doses of these compounds. The suitability of the method as a general technique for synthesis of different ether phosphoglycerides is discussed.

Animals↗
PubMed1994

Regulation of phosphatidic acid biosynthetic enzymes in Saccharomyces cerevisiae.

Phosphatidic acid is the biosynthetic precursor of all glycerolipids. To understand how phosphatidic acid biosynthesis is controlled in Saccharomyces cerevisiae, we studied the regulation of three enzyme activities involved in the synthesis of this glycerolipid precursor, i.e., glycerophosphate acyltransferase (GPAT), dihydroxyacetone phosphate acyltransferase (DHAPAT), and acyl DHAP reductase. GPAT activity was increased 3-fold, while DHAPAT activity was increased up to 9-fold in wild type cells grown in a nonfermentable carbon source compared to that of glucose-grown cells. The ratio of GPAT/DHAPAT activity was 12 in glucose-grown cells but only 4 in cells grown in glycerol/ethanol. In the previously characterized tpa1 mutant, (T. S. Tillman and R. M. Bell. 1986. J. Biol. Chem. 261: 9144-9149), GPAT was decreased 2-fold and DHAPAT 27-fold compared to activities in the wild type. Acyl DHAP reductase activity in both wild type and tpa1 cells grown on a nonfermentable carbon source was increased approximately 2-fold over that of glucose-grown cells. All three enzymatic activities increased as wild type cells grown on glucose entered the stationary phase of growth. Therefore, GPAT, DHAPAT, and acyl DHAP reductase activities appear to be regulated by the respiratory state of the cell. None of the activities was affected to a great extent by inositol, which is a key regulator of many enzymes involved in the synthesis of PtdOH-derived phospholipids in S. cerevisiae, nor by deletion of the mitochondrial genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗
PubMed1993

Purification of dihydroxyacetone phosphate acyltransferase from guinea pig liver peroxisomes.

Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), a peroxisomal enzyme which initiates the biosynthesis of glycerolipids (especially the ether-linked glycerolipids) in higher eukaryotes, has been purified by over 3250-fold from guinea pig liver. Initial stages of purification entailed isolation of liver peroxisomes by a combination of differential and density-gradient centrifugation. Dihydroxyacetone phosphate acyltransferase was solubilized from peroxisomal membranes with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate at moderate ionic strength (0.15 M NaCl). The solubilized enzyme was further purified by a regimen of size-exclusion chromatography, cation-exchange chromatography, and hydroxylapatite chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions during the purification of the enzyme, a 69-kDa protein band copurified with the enzyme activity, indicating that the monomeric enzyme may have a M(r) of 69,000. This was verified by further purifying the enzyme by chromato-focusing, when a single 69-kDa band was observed on SDS-PAGE. The M(r) of dihydroxyacetone phosphate acyltransferase determined by gel filtration is 90 kDa. The Vmax of the purified enzyme was approximately 4 mumol acyldihydroxyacetone phosphate (acylDHAP) formed per minute per milligram protein and the Km(DHAP) is approximately 70 microM when assayed at saturating concentrations of palmitoyl-CoA. Free coenzyme A inhibits the acyltransferase reaction with an inhibition constant (Ki) of approximately 0.76 mM. To date, this is the most highly purified DHAP acyltransferase (> 3200-fold) of mammalian origin.

Acyltransferases↗